Suppression of autoimmune diabetes by a plant-delivered cholera toxin B subunit-human glutamate decarboxylase fusion protein

We have generated transgenic potato plants synthesizing human glutamate decarboxylase (GAD65), a major autoantigen involved in the progression of insulin-dependent diabetes mellitus (IDDM). To target the plant synthesized autoantigen to enterocytes in the gut, a fusion gene was constructed in which the GAD protein was fused to the C-terminus of the cholera toxin B subunit (CTB) carrier peptide. To facilitate sequestration and pentamerization of the CTB fusion protein within the plant endoplasmic reticulum, a microsomal retention signal (SEKDEL) was linked to the C-terminus of the GAD protein. A flexible hinge peptide (GPGP) was inserted between the CTB and GAD moieties of the fusion protein to minimize stearic hinderance. The plant synthesized CTB-GAD fusion protein displayed the predicted pentamer molecular weight, G(M1)-ganglioside binding affinity, and native antigenicities of both CTB and GAD. Oral administration of transgenic potato tissues containing the CTB-GAD fusion protein induced both intestinal (IgA) and serum (IgG) anti-CTB antibodies in nonobese diabetic (NOD) mice. Pancreatic islet lymphocyte infiltration (insulitis) and clinical diabetes symptoms were significantly reduced in mice fed CTB-GAD containing potato tissues. However, mice fed transgenic potato tubers synthesizing only GAD65 protein showed a measurable but insignificant reduction in insulitis and diabetic symptoms confirming the importance of the CTB carrier protein in enhancement of the mucosal immune response at low autoantigen concentrations. The experimental results indicate that autoantigen-CTB carrier protein fusions, can make food plants effective production and delivery vehicles for immunotolerization against autoimmune disease.